Regulation of Alternative Splicing by the Core Spliceosomal Machinery

Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Regulation of alternative splicing by the core spliceosomal machinery Arneet L. Saltzman,1,2 Qun Pan,1 and Benjamin J. Blencowe1,2,3 1Banting and Best Department of Medical Research, The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada; 2Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP (small nuclear ribonucleoprotein) protein SmB/B9 self-regulates its expression by promoting the inclusion of a highly conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B9 in human cells results in reduced levels of snRNPs and a striking reduction in the inclusion levels of hundreds of additional alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA-binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors. [Keywords: alternative splicing; Sm proteins; snRNP; autoregulation; NMD; exon network] Supplemental material is available for this article. Received October 20, 2010; revised version accepted January 4, 2011. The production of multiple mRNA variants through indicated that the levels of some of these core splicing alternative splicing (AS) is estimated to take place in components can affect splice site choice. Microarray transcripts from >95% of human multiexon genes (Pan profiling revealed transcript-specific effects on splicing et al. 2008; Wang et al. 2008). AS represents a mechanism in yeast strains harboring mutations in or deletions of for gene regulation and expansion of the proteome. The core splicing components (Clark et al. 2002; Pleiss et al. most widely studied trans-acting factors regulating AS 2007). Knockdown of several core splicing factors in are proteins of the SR (Ser/Arg-rich) and hnRNP (hetero- Drosophila cells resulted in transcript-specific effects on geneous ribonucleoprotein) families, as well as numerous AS reporters (Park et al. 2004). Deficiency of the snRNP tissue-restricted AS factors (for review, see Chen and assembly factor SMN (survival of motor neuron) in a Manley 2009; Nilsen and Graveley 2010). These factors mouse model of spinal muscular atrophy (SMA) resulted generally regulate AS by recognizing cis-acting sequences in tissue-specific perturbations in snRNP levels and in exons or introns and by promoting or suppressing the splicing defects (Gabanella et al. 2007; Zhang et al. 2008). assembly of the spliceosome at adjacent splice sites. In Tiling microarray profiling analysis of fission yeast RNA contrast to these AS regulatory factors, much less is also revealed transcript-specific splicing defects of a tem- known about how or the extent to which components perature degron allele of SMN, and that some of the de- of the basal or ‘‘core’’ splicing machinery modulate splice fects could be alleviated by strengthening the pyrimidine site decisions. tract upstream of the branchpoint (Campion et al. 2010). The spliceosome is a large RNP complex that carries However, the features that underlie the differential sensi- out the removal of introns from pre-mRNAs. It comprises tivity of introns or alternative exons to particular defects the U1, U2, U4/6, and U5 small nuclear RNPs (snRNPs) in the core splicing machinery are not well understood. and several hundred protein factors (for review, see Wahl Many splicing regulatory factors can regulate the AS of et al. 2009). Studies in yeast and metazoan systems have their own pre-mRNAs (autoregulation), and in some cases have been observed to affect the AS of pre-mRNAs encoding other AS factors (for review, see Lareau et al. 2007a; McGlincy and Smith 2008). Such autoregulation 3Corresponding author. E-MAIL [email protected]; FAX (416) 946-5545. and cross-regulation are important for the establishment Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.2004811. and maintenance of AS factor levels across different GENES & DEVELOPMENT 25:373–384 Ó 2011 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/11; www.genesdev.org 373 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Saltzman et al. tissues and developmental stages. Autoregulation and when NMD is disrupted, as well as when expression of cross-regulation of AS factors can produce protein isoforms SmB is increased exogenously (Saltzman et al. 2008; this with different functional properties (Dredge et al. 2005; study). These results suggested that AS-NMD plays a role Damianov and Black 2010), and have also been shown in in the homeostatic regulation not only of AS regulatory many cases to regulate gene expression by producing factors, but of components of the basal splicing machin- alternative transcripts containing premature termina- ery as well. Moreover, the results further suggested that tion codons (PTCs) that are degraded by nonsense-me- core spliceosomal components may regulate specific AS diated mRNA decay (AS-NMD). Consistent with their events in addition to their well-studied critical roles in functional importance, regulated AS events involved in constitutive splicing. AS-NMD of splicing factors often lie within highly con- In the present study, we investigated the role of the core served or ultraconserved sequence regions (Lareau et al. spliceosomal machinery in AS, focusing on a detailed 2007b; Ni et al. 2007; Yeo et al. 2007; Saltzman et al. investigation of SmB/B9. Our results confirm that SmB/B9 2008). functions in homeostatic autoregulation through the Using AS microarray profiling following knockdown inclusion of a highly conserved PTC-introducing exon of NMD factors, we previously identified highly con- in its own pre-mRNA. This mode of regulation depends served, PTC-introducing alternative exons in genes en- on a suboptimal 59ss associated with the SNRPB PTC- coding multiple core splicing factors (Saltzman et al. introducing exon and appears to be controlled by changes 2008). These genes included SNRPB, which encodes in the level of U1 snRNP as a consequence of SmB/B9 the core snRNP component SmB/B9, a subunit of the depletion. We also show that knockdown of SmB/B9 leads heteroheptameric Sm protein complex that is assem- to a striking reduction in the inclusion levels of many bled by the SMN complex onto the Sm-site of U1, U2, U4, additional alternative exons, which are significantly and U5 snRNAs (for review, see Neuenkirchen et al. enriched in functions related to RNA processing and 2008). The SmB and SmB9 proteins are nearly identical RNA binding. Changes in the inclusion levels of a subset and arise from alternative 39 splice site (ss) usage in the of these alternative exons also appear to control expres- terminal exon of SNRPB (Fig. 1A, top; Supplemental Fig. sion levels of the corresponding mRNAs by AS-NMD. 1), leading to an additional repeat of a short proline-rich Our results thus reveal an important role for the core motif at the C terminus of SmB9 (van Dam et al. 1989). spliceosomal machinery in establishing the inclusion The PTC-introducing alternative exon in SNRPB lies levels of a specific subset of alternative exons, and further within a region of the second intron that is highly suggest that changes in the levels of these alternative conserved in mammalian genomes (Fig. 1A). Splice var- exons control the expression of other RNA processing iants including this PTC-introducing exon accumulate factors. Figure 1. The inclusion of a highly conserved PTC-introducing alternative exon in SNRPB is affected by SmB/B9 knockdown. (A, top panel) Diagram of the exon/intron structure of the SNRPB gene. The two encoded proteins, SmB and SmB9, arise from alternative 39 ss usage in the final exon. A conservation plot (phyloP), is shown below (generated using the University of California at Santa Cruz genome browser (http://genome.ucsc.edu; Rhead et al. 2010). (Bottom panel) An expanded view of the region between the second and third SNRPB exons. The highly conserved area within this intron contains an alternative exon (‘‘A’’). This alternative exon and its conserved flanking intron regions (boxed in blue) were cloned into a mini- gene (miniSmB) in which they are flanked by heterologous intron and exon sequences. (B) RT–PCR assays confirm an increase in the level of the PTC-containing SNRPB variant when NMD is abrogated by knockdown of UPF1. See also Supplemental Figures 2 and 3. (C) Knock- down of SmB/B9 leads to more skipping of the SNRPB alternative exon in miniSmB. HeLa cells were transfected with a control nontargeting (NT) siRNA, or an siRNA targeting the 39 untranslated region (UTR) of SmB/B9. Cells were then cotransfected with miniSmB and either an empty vector or a vector encoding a 3xFlag-tagged cDNA, as indicated above the gels. (Top panel) To assay alternative exon inclusion in miniSmB, RT–PCR assays were performed using primers specific for the flanking

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